The broad objective of the proposed research is to develop and exploit nuclear magnetic resonance (NMR) techniques for the study of protein structure and function in biological membranes. We have prepared six different cytochrome c derivatives in each of which a single lysine group has been specifically trifluoroacetylated. All surface areas on cytochrome c have been labelled with F-19 NMR probes. F-19 methods will be used to accurately locate the binding sites on cytochrome c for cytochrome oxidase, cytochrome reductase, and phospholipid membranes. We will investigate possible conformational changes at these binding sites due to electron transfer, and also attempt to measure distances between the F-19 labels on cytochrome c and paramagnetic metals in the oxidase and the reductase. This information is very important for an understanding of what types of electron transfer mechanisms might be feasible. F-19 NMR methods will be used to study the interaction of the cytochrome c derivatives with intact mitochondria. These studies should allow us to determine what components of the mitochondrial membrane cytochrome c is binding to under different physiological conditions. A central question is whether cytochrome c can bind to cytochrome oxidase and cytochrome reductase simultaneously, thus transporting electrons directly, or whether the binding sites are competitive and cytochrome c must undergo rotational and translational motion to transport an electron from cytochrome reductase to cytochrome oxidase.